Method and device for determining biological analytes

ABSTRACT

The invention relates to a method for quantitatively determining biological analytes in an aqueous solution in the presence of one or more functionalised surfaces, wherein the aqueous solution comprises at least one type of biological analyte and at least one type of fluorescene marker, characterised in that the quantity and/or concentration of the biological analyte or analytes is determined by measuring the florescence emission of the unbound fluorescence markers, as well as to a device for carrying out said method.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/124,076, filed 7 Sep. 2016, which is a 371 National Stage Applicationof PCT/EP2015/054671, filed 5 Mar. 2015, which claims priority to DE14158692.5, filed 10 Mar. 2014, the contents of which are incorporatedherein by reference in their entireties.

The application relates to a method for qualitatively and/orquantitatively determining biological analytes by measuring theluminescence of the unbound luminescence markers, as well as to a devicetherefor and to the use of the device.

The quantitative and/or qualitative determination and the counting ofbiological units such as, for example, nucleic acids, proteins,antibodies, bacteria, viruses or cells, what are known as biologicalanalytes, is an important method in the development of modern activeingredients, in other research in the field of the life sciences and inthe production of biopharmaceuticals.

The demands made of a method or process for determining biologicalanalytes are generally high and vary greatly, in particular as regardsconcentration ranges, accuracy of measurement and sample preparation.Even when determining the same biological analytes, the measuringmethods can vary greatly depending on the purpose of the measurement. Indiagnostics, for example, a protein is measured in a very lowconcentration in blood serum samples, while in biotechnologicalproduction methods proteins are situated in a culture medium, where theyare present in concentrations which are several powers of ten higher.Furthermore, the methods can be such that, as well as determining theanalytes, they are also able to provide further information, for exampleabout the activity or the affinity and the specificity. Theenzyme-linked immunosorbent assay, abbreviated as “ELISA assay” or“ELISA method”, has for decades been established as the standard methodfor determining particular biological analytes, in particular proteinsand antibodies. It is an immunoassay to which an enzymatic colourreaction is coupled. The principle of the ELISA assay consistssubstantially in binding the biological analyte (for example a protein)to a surface (for example a microplate) by catcher molecules. Thecatcher molecules are typically antibodies, fragments thereof or alsoprotein A or protein G.

In the widely used sandwich ELISA method, an antibody is used as thecatcher and then a detection antibody is bound to the analyte at asecond binding site. An enzyme (for example horseradish peroxidase, HRP)is coupled to the detection antibody, which enzyme, by addition of asubstrate for the enzyme, initiates an enzymatic reaction, by means ofwhich dyes form. Typical substrates are tetramethylbenzidine (TMB) ordiaminobenzidine (DAB), which colour the solution blue or brown. Beforethe colouring can be measured by spectrometry, the enzymatic reactionmust be stopped by adding a stop reagent. The amount of dye produced bythe HRP is proportional to the amount of analyte and is then measuredwith a suitable detection device, generally a UV/VIS photometer. Alsoknown in this context are enzyme substrates which yield a fluorescentproduct (for example resorufin), which is detected with a fluorescencemeter. By means of the enzymatic reaction, a large number of dyemolecules can be produced in the sandwich ELISA assay for each boundanalyte molecule. As a result of this enhancement effect, the sandwichELISA assay is generally very sensitive.

Also common are ELISA methods which are characterised in that theenzyme-conjugated detection antibody is bound not directly to theanalyte but to a further primary antibody which in turn bindsspecifically to the analyte. This variant is employed ifenzyme-conjugated primary antibodies are not available and is overallmore sensitive for non-specific binding of the antibodies with oneanother.

The direct ELISA methods are distinguished in that there is used as thecatcher molecule not an antibody but an antigen, which is bound by theantibody to be measured. Detection takes place analogously to themethods described above via detection antibodies.

A disadvantage of the ELISA method is that it involves a large number ofincubation steps and washing steps, which often have to be carried outrepeatedly. These working steps are not or are only partiallyautomatable and are therefore predominantly carried out manually atpresent. This makes the ELISA assay intensive in terms of personnel andtime. In the ELISA method, test times of from 4 to 5 hours are oftenrequired, so that the ELISA assay cannot be used in processes in which aquick result is required or a large number of samples are to bemeasured, for example in HTS (high throughput screening).

A further disadvantage of the ELISA method is that the large number ofworking steps gives rise to a relatively large total error, since eachof the working steps causes its own variance and those variances add upto the total variance.

Attempts have already been made to make the ELISA method quicker andless intensive in terms of personnel and cost. One modification of theELISA method is the “fluorescent immunoassay” (FIA). Fluorescentimmunoassays are generally simpler to carry out than the above-mentioned“conventional” ELISA method.

By using a detection antibody labelled with a fluorescent dye, it ispossible in the fluorescent immunoassay to work without an enzymaticreaction. The number of method steps is thereby reduced, and deviationsbased on fluctuating enzyme activity are thereby eliminated. Fluorescentimmunoassays can be carried out in homogeneous or heterogeneous systems.In heterogeneous systems, special particles are often used and thefluorescence at the particles is measured, while in homogeneousfluorescent immunoassays the fluorescence is measured in solution.Several steps of the conventional ELISA method are thus saved, and thetest time is reduced to usually from 2 to 3 hours. However, dispensingwith the enzyme reaction often has the result that the sensitivity inthese methods is lower than in the ELISA.

An example of a homogeneous fluorescent immunoassay is the Delfiatechnology (dissociation-enhanced lanthanide fluorescent immunoassay)from Perkin-Elmer. In the method, europium complexes bound to thedetection antibody are brought into solution and then measured withfluorescence, in particular time-resolved fluorescence. A further knownmethod is the TR-FRET technology, which is offered by Cisbio as HTRFtechnology (homogeneous time resolved fluorescence), and the LANCEtechnology (lanthanide chelate excite) from Perkin-Elmer.

The above-mentioned TR-FRET method is based on the principle that twoantibodies binding one another are so labelled with fluorescent dyesthat the dyes are able to carry out a fluorescence resonance transfer(FRET) when they are brought into spatial proximity, that is to say whenthey are bound to one another. A FRET donor is bound to one antibody anda FRET acceptor is bound to the other antibody. FRET only works when thedistance between the antibodies is less than 10 nm. If theabove-mentioned antibodies bind to one another, the FRET donor can beexcited to fluorescence with light of a specific wavelength. By means ofthe emitted light of the FRET donor, the FRET acceptor is excited tofluorescence emission, and this is measured. That is to say, thefluorescence emission of the FRET acceptor can only be measured if thetwo antibodies (in the solution) are bound to one another. If such asolution is placed in a vessel and an analyte which binds to the FRETacceptor or the FRET donor is added thereto, one of the two FRETpartners is displaced from the complex, so that the fluorescenceemission of the FRET acceptor diminishes in dependence on theconcentration of the analyte.

FRET is a technique which is widely used in research to detect thebinding of two molecules. However, it is also known that the efficiencyof the transfer is greatly dependent on the composition of the sample.In order to make a routine method out of this technique, special FRETdonors consisting of a complex between a europium or terbium ion and amacrocycle (for example based on tris-bipyridines) are used in TR-FRETtechnology. These donors have the property of emitting longer-lastingfluorescence in comparison to other fluorescent substances which may bepresent in the sample. The fluorescence measurement therefore takesplace only in a defined time interval after a flash of light. As aresult, a period of time is allowed to elapse until the interferingfluorescence from the sample has subsided and only the FRET fluorescencestill occurs. Nevertheless, both the emission of the FRET donor and thatof the FRET acceptor must be measured in TR-FRET technology, and theratio thereof must be determined so that matrix influences and quenchingof the fluorescence emission can be compensated for mathematically. Inorder for this compensation to function optimally, the two emissionwavelengths must be measured simultaneously and not sequentially. Thismeans that the measuring devices for accurate TR-FRET measurements mustbe equipped with two detectors. Typical measuring ranges for the TR-FRETmethod are approximately from 10 to 5000 ng/ml of antibody.

A further modification of the conventional ELISA assay is the AlphaLISAmethod from Perkin-Elmer. Here, a pair of antibodies labelled withprobes is brought into spatial proximity by binding to different bindingsites of the same analyte.

In contrast to TR-FRET, the probes used are not fluorophores having alow molecular weight but donor and acceptor particles (donor andacceptor beads) having a size of approximately from 250 to 350 nm. Byirradiating the donor beads with laser light having a wavelength of 680nm, singlet oxygen is released therefrom and triggers light emissionhaving a wavelength of approximately 615 nm at the acceptor beadsituated in proximity. The light emission is then measured and serves toquantify the analyte. Unlike in TR-FRET, the molecules carrying probescannot be added simultaneously in the AlphaLISA, so that a secondreaction time (incubation time) is necessary and the protocol isconsequently lengthened.

The donor beads are light-sensitive. It is possible to work with themonly in the dark or in green light, which represents a considerabledisadvantage of the method. The laser to be used in the AlphaLISA assayis often only incorporated into special fluorescence plate readers,which makes the test expensive since it is necessary to have or purchasethe devices.

In particle-supported heterogeneous fluorescent immunoassays, as in theconventional ELISA methods, the biological analyte is first bound to thesurface, in this case to functionalised insoluble particles, by catchermolecules. After a washing step, fluorescence-labelled detectionantibodies are added, the whole is mixed, and then the unboundfluorescence-labelled detection antibodies are removed. The quantity offluorescence-labelled detection antibodies bound to the particles ismeasured, the content of the biological analyte being determined via thefluorescence of the fluorescence markers bound to the particles. Such amethod, or determination, is known (see overview article by C. F.Woolley and M. A. Hayes, “Recent developments in emergingmicroimmunoassays”, Bioanalysis (2013) 5(2)).

In the “GYROS technology” used by Gyros AB, microfluidic structures areused to centrifuge small sample quantities onto particle beds (usuallyin the form of small columns), on which there form fluorescence-labelledsandwich complexes, which are measured. The entire microfluidic systemis produced in round plastics discs, the liquids (for example dissolvedcatcher molecules, sample, washing solutions) being forced outwardsthrough the channels by the rotation of the disc (as in the case of aCD-ROM) about its central axis. Since GYROS technology can only be usedto measure concentrations of greater than or equal to 1 ng/ml, it isused above all in biotechnological process development and control.

Quanterix offers a technology by the name of SIMOA (single moleculearray), in which sandwich immunocomplexes are formed on particles. Theparticles are then distributed microfluidically on a chip to hundreds ofthousands of cavities (wells/indentations), each of which is able toreceive only one particle. For the detection, the cavities are thenclosed and an enzyme is activated, which produces a fluorescent dye. Atvery low concentrations of analyte, there is on average less than oneanalyte molecule in a cavity, that is to say there are only cavitieswith or without fluorescence, which are then counted. This assay istherefore also referred to as “digital ELISA”. Detection limits below10⁻¹⁵ M can be achieved therewith (see Rissin et al.: “Single-moleculeenzyme-linked immuosorbent assays detects serum proteins atsubfemtomolar concentrations”, Nature Biotechnology, 28(6), 595).

GYROS and SIMOA are multi-stage processes, and complex microstructuredconsumables (such as, for example, the discs mentioned above) as well asdedicated and expensive readers are required.

For measuring fluorescence-labelled immunocomplexes which are bound toparticles, it is also possible to use flow cytometers. Here, theparticles are transported individually through a flow system to ameasuring point at which they are excited to fluorescence by laserlight. Measurement with a flow cytometer has the advantage thatdifferent analytes can be determined at the same time. The particlesused for that purpose carry a colour coding on the inside, that is tosay are doped with differentiable fluorescent dyes, each of the coloursrepresenting binding to a specific analyte. For the measurement, twolasers are used simultaneously. The first laser identifies the particletype via the colour coding, and the second laser identifies the contentof bound immunocomplex. In XMAP technology (Luminex Corp.), up to onehundred different analytes can be detected in one test procedure; thisis referred to as multiplex applications.

In addition to the above-mentioned methods, further measuring methodsare used, in particular in the field of process development and controlfor producing recombinant proteins and antibodies, which are lesscomplex than ELISA, for example biolayer interferometry (BLI, from PallForteBio) or surface plasmon resonance (SPR, GE Healthcare, Biacore).These methods, in particular BLI, permit a higher degree of automationand are based on the binding of the analyte to surfaces which aretypically coated with protein A or G or with antibodies.

Binding the analytes to the surface brings about a change in the opticalproperty of the surface, which change is measured. The quantity ofmolecules bound to the surface is calculated therefrom. Thus, measuringranges of from 0.5 to 2000 μg/ml are achieved with BLI and measuringranges of from 0.15 to 10 μg/ml are achieved with SPR. A disadvantage ofthis method is that comparatively expensive and specialised measuringdevices are required, as are special consumables (for example sensorsand chips).

All the methods mentioned above are disadvantageous as regards theircomplex test procedure. They often require many working steps for thequantification of biological analytes and are therefore complex toperform. Likewise, there are applications and scientific problems inwhich it is desirable to have available a simplified method, based on animmunoassay, with which more highly concentrated samples can be measuredand/or with which results are obtained quickly. It is further desirableto have available an immunoassay with which special antibodies can bedetermined in diverse liquids, such as, for example, serum or cellculture supernatants. This is an advantage in particular in the case ofthe recombinant (technical) production of antibodies in order to be ableto make qualitative and/or quantitative statements simply, quickly andinexpensively during the production of proteins. It is further desirableto have available a method, in particular an immunoassay, fordetermining biological analytes which requires only a small number ofworking steps, whereby time and reagents can be saved. There istherefore a need for a simple and rapid method for determiningbiological analytes, preferably in the form of an immunoassay, which issimple to carry out and avoids all or at least some of the disadvantagesmentioned above.

Accordingly, the object of the present invention is to provide a methodwith which all or at least some of the above-mentioned disadvantages areavoided.

The object has been achieved by the method described in the claims, inparticular by the device according to the invention and the use thereof.The important factor is that the luminescence or fluorescence emissionof the unbound luminescence or fluorescence markers is measured, so thatbiological analytes can be determined simply, quickly and inexpensively.

Accordingly, the invention relates to a method for determining(especially for quantifying) biological analytes in an aqueous solutionin the presence of one or more functionalised surfaces, wherein theaqueous solution comprises at least one type of biological analyte andat least one type of luminescence marker, preferably fluorescencemarker, characterised in that the quantity and/or concentration of thebiological analyte or analytes is determined by measuring theluminescence emission, preferably fluorescence emission, of the unboundluminescence markers, preferably fluorescence markers.

Any desired assays can be carried out with the method according to theinvention. The method according to the invention is preferably animmunoassay, in particular an immunoassay selected from a groupconsisting of direct immunoassay, sandwich immunoassay, displacementimmunoassay (also called inhibition assay herein), competitiveimmunoassay and secondary immunoassay.

In one embodiment [V-1], there are used as the functionalised surfacesin the method according to the invention functionalised particles ofpolymer or a polymer mixture, on the surface of which particles thereare catcher molecules which bind to the biological analyte or analytesand/or to the fluorescence marker or markers. The functionalisedparticles advantageously have a mean diameter in the range of fromapproximately 20 to approximately 200 μm or in the range of fromapproximately 80 to approximately 200 μm.

In one embodiment [V-2], functionalised magnetic particles are used asthe functionalised surfaces in the method according to the invention.The magnetic particles advantageously have a mean diameter in the rangeof from approximately 1 to approximately 100 μm. Magnetic particles haveferromagnetic or superparamagnetic properties. They conventionallycomprise a magnetic core of ferrite or magnetite, which is surrounded bya shell of polymers or polymer mixtures. They are functionalisedaccording to the non-magnetic particles, that is to say equipped withcatcher molecules.

In one embodiment [V-3], the method according to the invention comprisesthe following steps:

(a) introducing at least one type of functionalised particles orfunctionalised magnetic particles into a measuring chamber which has adetection region, which is accessible to light through the bottom of themeasuring chamber, and a separation region which is not accessible tolight;

(b) introducing a sample comprising at least one type of biologicalanalyte into the measuring chamber;

(c) introducing at least one type of fluorescence marker into themeasuring chamber;

(c′) mixing the introduced particles, sample and fluorescence markers inthe measuring chamber;

(c″) separating the unbound fluorescence markers from bound fluorescencemarkers (preferably by sedimentation or centrifugation), so that thebound fluorescence markers are in the separation region;

(d) measuring the fluorescence emission of the unbound fluorescencemarkers in the detection region: and

(e) determining the quantity and/or concentration of the biologicalanalyte or analytes.

Measuring chamber is here also understood as being a microplate wellthat is equipped according to the invention. The method according to theinvention is preferably carried out with microplates whose wells areequipped as measuring chambers according to the invention.

Steps (a), (b) and (c) can be carried out before or after a furthermethod step, wherein the further method step comprises introducingfurther test components or substances, such as, for example, primaryantibodies or auxiliary substances, such as detergents, into themeasuring chamber.

Steps (a), (b) and (c) can take place sequentially, or at least two ofthe steps take place simultaneously.

In a variant [V-4] of the method according to the invention, step (a)and/or step (c) is carried out before the remaining method steps,preferably with a large time interval relative to the remaining steps.

In a variant [V-5] of the method according to the invention, thefunctionalised particles are magnetic (magnetisable).

In a further variant [V-6], functionalised magnetic particles are usedand step (c″) is carried out by sedimentation and by applying atemporary or permanent magnetic field.

In one embodiment [V-7] of the method according to the invention, afluorescence microscope is used for measuring the fluorescence emissionof the unbound fluorescence markers in step (d). In the devices,measuring chambers or microplates according to the invention which areto be used, the opaque layer on the bottom can then be omitted. Thefluorescence microscope must be so adjusted that it measures only thefluorescence emission that occurs in the detection region, while thefluorescence emission coming from the separation region is masked out.

Microplates are known. They contain a plurality of cavities (also calledwells, cups, indentations or recesses) which are isolated from oneanother. The number of cavities on a microplate can vary. The followingarrangements are available commercially: 2×3 (6 wells), 4×3 (12 wells),4×6 (24 wells), 6×8 (48 wells), 8×12 (96 wells), 16×24 (384 wells),32×48 (1536 wells). The bottoms of the cups of the commerciallyavailable microplates can have different shapes. The following bottomsare available commercially: F-bottom (flat bottom), C-bottom (flatbottom with minimally rounded corners), V-bottom (conically taperingbottom) and U-bottom (U-shaped well). Microplates having an F-, C- orU-bottom, into which the device is introduced, are particularly suitableaccording to the invention.

Particle-supported methods for determining biological analytes are known(see U.S. Pat. No. 4,731,337B, DE 102004038163A, WO 86/04684, WO94/29722, U.S. Pat. No. 4,115,535B, WO 2011/045022). These methods areall based on measuring the fluorescence of bound fluorescence markers.

Devices for separating insoluble constituents from aqueous solutions areknown. WO 2011/031236 and US 2010/0028935, for example, describespecially shaped devices for separating corpuscular particles from anaqueous solution in order thus to increase the accuracy of measurementin transmission spectroscopy and absorption spectroscopy. However, thesedevices are not suitable for use in the method according to theinvention since it is not possible with those devices reliably tomeasure the luminescence of unbound luminescence markers, or thefluorescence of unbound fluorescence markers, in order to quantify thebiological analyte or analytes to be determined.

The method therefore also provides special devices with which the methodaccording to the invention can be carried out.

Accordingly, the invention relates also to a device [0] for determiningbiological analytes by measuring the luminescence of the unboundluminescence markers, in which bound and unbound luminescence markers inan aqueous solution are spatially and optically separated from oneanother using one or more functionalised surfaces, wherein the devicehas for the separation an at least partly transparent structuralelement, wherein the base of the device, apart from the base area of thestructural element, is opaque and the aqueous solution comprises atleast one type of biological analyte and at least one type ofluminescence marker, and wherein at least one type of biological analyteand optionally at least one type of luminescence marker binds to one ormore functionalised surfaces.

The invention relates further to a measuring chamber for use in themethod according to the invention, in which functionalised magneticparticles are used. The bound luminescence markers, preferablyfluorescence markers, are separated from the unbound luminescencemarkers by directed sedimentation with the aid of an opaque magneticelement placed beneath the measuring chamber. The magnetic element hasapertures which are so arranged that measuring windows are formed on thebottom (base) of the measuring chamber. The magnetic element can bepermanently connected to the base, or it is removable. The measuringchamber does not have a structural element.

By means of the magnetic field, the sedimentation of the functionalisedmagnetic particles is so guided that no particles settle above themeasuring window. The luminescence, preferably fluorescence, is measuredwith a fluorescence meter through the measuring window in the bottom ofthe measuring chamber.

The invention relates further to the use of the above-mentionedmeasuring chamber, or of a microplate that is equipped with at least onesuch measuring chamber, in the method according to the invention,wherein functional magnetic particles are likewise used.

In one embodiment [A] of the device [0] according to the invention, oneor more functionalised surfaces in the form of functionalised particlesare introduced into the device according to the invention, wherein thebound luminescence markers are situated in the separation region of thedevice and the unbound luminescence markers are situated in thedetection region, wherein they are preferably homogeneously distributedtherein.

In one embodiment [B] of the device [0] according to the invention,there are one or more functionalised surfaces inside the device,preferably in the region of the device that is situated beneath the endof the structural element remote from the base, preferably that issituated on the base of the device. Separation takes place by binding ofthe biological analyte and the luminescence marker to one or more of theoptionally immobilised functionalised surfaces situated in the device.

In a further embodiment [C], the invention relates to a device, asdescribed above or in embodiment [A] or [B], in which the structuralelement is in the form of a protrusion (preferably in the form of anupwardly tapering protrusion), the cross section of which can have anydesired geometry (for example circular, rectangular, triangular),wherein the end of the structural element that is remote from the baseof the device is such that no test components, in particular noparticles, settle there. It can be flat, for example, or have a convexshape and/or have a small diameter.

The structural element situated in a measuring chamber can have theshape of a mandrel, cone, truncated cone, pyramid or truncated pyramid,the base area of which is n-cornered, where n represents an integer inthe range of from 3 to 10, preferably 3, 4, 5, 6, 7 or 8. It can furtherhave a shape which is derived from a cone or a pyramid, for example around-based cone or a 4-sided square-based pyramid.

The structural element can further have an optical component, forexample a lens, at the end that is remote from the base of the device.

The structural element is transparent. It is made of a suitabletransparent material or material mixture. It is important that thematerial does not have any autofluorescence which interferes with themeasurement. Transparent materials are known and can easily be selectedby a person skilled in the art. Such materials are, for example,polystyrene, COC (cycloolefin copolymer), polypropylene or polymethylmethacrylate (PMMA). Polypropylene and PMMA are very suitable.Polystyrene, in particular when it is processed while hot, has a certaindegree of autofluorescence (Young et al., Anal Chem. 2013 January 2:85(1): 44-49) and is therefore not preferred according to the invention.

The material or material mixture can be used in a thickness (layerthickness) of ≤1 mm. Preferred thicknesses are in the following ranges:from approximately 0.05 to approximately 0.5 mm, from approximately 0.1to approximately 0.45 mm, from approximately 0.15 to approximately 0.4mm, from approximately 0.2 to approximately 0.4 mm, or fromapproximately 0.2 to approximately 0.35 mm.

In a further embodiment [D], the invention relates to a device, asdescribed above or in one of embodiments [A], [B] or [C], in which thereis a separation region and a measurement region (detection region),wherein the measurement region has a transparent measuring window or isoptically connected to such a measuring window.

In a further embodiment [E], the invention relates to a device, asdescribed above or in one of embodiments [A], [B], [C] or [D], in whichthe base of the structural element is in the form of a measuring window.The luminescence is then measured by means of a luminescence meter,preferably a fluorescence meter. The excitation and the detection of theemission take place from beneath the device.

When the base and the end of the structural element that is situated ina device or measuring chamber are optically connected to one another,excitation light irradiated in from the base of the device passesthrough the structural element into the detection region, where itexcites the unbound luminescence markers, preferably fluorescencemarkers, to emission, which is then measured likewise from the base ofthe device. The end of the structural element and the base then togetherform the measuring window. When selecting a suitable shape for thestructural element, it must be ensured that particles do not interferewith the excitation and emission light. It is therefore particularlyadvantageous if the end of the structural element has a convex shape andthe structural element is in the form of a protrusion which tapersupwards. If the end is flat, it is advantageous if the surface is smalland/or the surface is such that no particles or only a non-interferingnumber of particles (also referred to in the present case as“substantially no”) are able to settle. In the case of flat ends too, itis advantageous if the protrusion tapers towards the end that is remotefrom the base.

In a further embodiment [F], the invention relates to a microplate ineach of the recesses, cups or wells of which there is integrated adevice as described above or in one of embodiments [0], [A], [B], [C],[D] or [E].

Accordingly, the invention relates in one embodiment [F-1] to amicroplate for carrying out the method according to the invention,characterised in that there is introduced into at least one well of themicroplate a device which has an at least partly transparent structuralelement which is in the form of an upwardly tapering protrusion and thecross section of which can have any desired geometry, wherein the end ofthe structural element that is remote from the base is such thatsubstantially no test components settle there, and wherein the base ofthe device, apart from the base area of the structural element, isopaque, and wherein the edges of the well form the side edges of ameasuring chamber.

In a further embodiment [F-2], the invention relates to the microplateas described in embodiment [F-1], characterised in that the originalbottom of the microplate is replaced by a one-piece base havingstructural elements, wherein a structural element is situated in each ofthe wells of the microplate, and wherein the base, apart from the basearea of the structural elements, is coated with an opaque layer.

In this case, the base is made of the same material as the structuralelements and the criteria mentioned in connection with the structuralelements as regards material properties and layer thickness apply forthe selection of the material from which the base is formed.

The invention relates also to the production of the microplate describedas embodiment [F-2], comprising the following steps:

-   -   (x) replacing the bottom of a microplate by a base having        structural elements, which base preferably has as many        structural elements as there are wells in the microplate;    -   (y) connecting the base to the microplate; and    -   (z) applying an opaque layer to the underside of the base,        wherein the base area of the structural element is kept free.

The connection of the base to the microplate and the application of anopaque layer take place as described below in the same context.

The one-piece base having structural elements can be produced by shapingmethods, for example by injection moulding methods, additive productionmethods (for example 3D printing) or thermoforming (hot forming, deepdrawing or vacuum forming).

It is of course possible that a structural element is not present ineach of the wells of a microplate. Likewise, it is possible thatdifferently shaped structural elements are present in the wells of themicroplate. A one-piece base having structural elements can thus havestructural elements of different shapes.

In a further embodiment [G], the invention relates to a measuringchamber containing a device as described above or in one of embodiments[0], [A], [B], [C], [D] or [E].

In a further embodiment [H], the invention relates to the use of thedevice, measuring chamber or microplate, as described above or in one ofembodiments [0], [A], [B], [C], [D], [E], [F] or [G], in an immunoassay,in particular direct immunoassay, sandwich immunoassay, competitiveimmunoassay or secondary immunoassay, wherein the luminescence markersare then preferably fluorescence markers, for qualitatively orquantitatively determining biological analytes by measuring theluminescence of the unbound luminescence markers.

In addition to methods described above, the invention relates further toa method for qualitatively and/or quantitatively determining biologicalanalytes using the device, measuring chamber or microplate according tothe invention, in particular as described above or in one of embodiments[0], [A], [B], [C], [D], [E], [F] or [G], which method comprises thefollowing steps:

-   (a) introducing at least one type of functionalised surface into the    device;-   (b) introducing a sample comprising at least one type of biological    analyte into the device;-   (c) introducing at least one type of luminescence marker into the    device;-   (d) measuring the luminescence emission of the unbound luminescence    markers; and-   (e) determining the quantity and/or concentration of the biological    analytes.

According to the invention, functionalised surfaces can already bepresent in the device, or they are introduced into the device alone ortogether with the measuring solution (sample) in the method according tothe invention.

In one embodiment of the invention, functionalised particles are used asthe functionalised surfaces.

In the method according to the invention, several types offunctionalised surfaces/particles can be present, or several types offunctionalisation can be present on a surface/particle surface. That isto say, the nature of the functionalisation of the surface can bedifferent. This has the result that several types of biological analytescan be measured by the method according to the invention and in thedevice and measuring chamber according to the invention or with themicroplate according to the invention. To that end, a distinctluminescence marker which is to be excited and/or emits with a differentwavelength must be used for each of the analytes.

If functionalised magnetic particles are used, the above appliescorrespondingly.

In connection with the present invention, the term “functionalised”means that catcher molecules (also called “catchers” hereinbelow) arepresent on a surface, preferably on a particle surface. For thefunctionalisation of surfaces or particle surfaces, the surface ispopulated with at least one type of catcher molecule. Catcher moleculesare molecules which bind physically or chemically to the biologicalanalyte or analytes and/or to the luminescence marker or markers. It isirrelevant whether the particles are magnetic or non-magnetic. Bothtypes of particles can be functionalised as described herein.

According to the invention, “functionalise” also means loading magneticor non-magnetic particles with suitable metal ions, as describedhereinbelow in connection with immobilised metal affinity chromatography(IMAC), so that the metal ion complexes formed (for example NTA-Ni²⁺)form the catcher molecules, which then bind test components that havethe corresponding tags.

For competitive assays, it is necessary for the functionalised surfaces,in particular particle surfaces, to have catchers to which both thebiological analyte and the luminescence marker can bind physicallyand/or chemically at the same binding site. For the remaining assays,the catchers are to be such that that they bind only the biologicalanalyte or analytes or, if desired, the luminescence marker or markers,preferably fluorescence markers.

The same applies if functionalised magnetic particles are used.

In a functionalisation variant, the catcher molecule has only bindingsites that are selected specifically for one type of biological analyte,while in another variant the catcher molecule has a plurality ofdifferent binding sites, each binding site being so selected that itbinds only a special biological analyte. With this variant, it ispossible that different types of biological analytes bind to thefunctionalised surface.

If different types of biological analytes are to be measured at the sametime, it is also possible in the method according to the invention touse differently functionalised (magnetic) particles in a measuringchamber.

One functionalisation variant comprises equipping the surfaces withdifferent types of catcher molecules, each of the catcher moleculeshaving at least one binding site for the biological analyte to bedetermined.

If functionalised particles or functionalised magnetic particles whichare capable of binding several types of biological analytes are used inthe method according to the invention, a distinct luminescence marker,preferably fluorescence marker, which is to be excited and/or emits witha different wavelength must be used for each of the analytes.

If the method according to the invention is to be carried out reliably,in particular when it is a direct (immuno)assay, sandwich (immuno)assayor secondary (immuno)assay, it is important that neither the catchermolecules nor the surfaces or particle surfaces bind specifically ornon-specifically to test components other than the biological analyte oranalytes. Exceptions are the competitive (immuno)assay and thedisplacement assay. In the competitive (immuno)assay, the catcher canbind the luminescence marker and the specific biological analyte at thesame binding site. In the displacement assay, the catcher binds theluminescence marker, preferably the fluorescence marker.

Catchers suitable for functionalising surfaces and particle surfaces areknown and are so selected that the desired test component or the desiredtest components bind to the catcher. Suitable catchers are mostlyproteins (for example antibodies) which are optionally bound to thesurfaces or particle surfaces by linker systems. The surface or particlesurface can be populated with catchers by covalent or non-covalentbinding. If one of the test components (for example the biologicalanalyte) is an antibody, then the catcher is usually a protein.

Known catchers for antibodies are, for example, protein A fromStaphylococcus aureus and protein G from streptococci.

The same applies if functionalised magnetic particles are used.

Examples of surfaces equipped with a linker system arestreptavidin-coated particles as are described in greater detailhereinbelow, for example Streptavidin Mag-Sepharose® particles (VWR,Art. No. 28-9857-38). A further example is agarose particles which carrynitriloacetic acid (NTA) or iminoacetic acid groups (IDA) on theirsurface, which particles form very stable complexes with metal ions andare suitable for binding to the His-tag labelled protein.

A common material consisting of porous material that is suitable forproducing functionalised particles is agarose, which is availablecommercially under the name Sepharose™ (GE Healthcare). Sepharose, oragarose, is obtainable in different degrees of crosslinking and withdifferent binding capacities. Prior to functionalisation, that is to sayloading with catchers, the agarose particles are activated with achemical in order to ensure efficient binding to catcher molecules. Asuitable chemical for the activation is CNBr.

As already described, common catcher molecules are proteins. In order tobind proteins to surfaces, in particular particle surfaces, it ispossible to use chemicals which function as linkers between the surfaceand the catcher molecule (for example streptavidin or homologues thereof(for example avidin, neutravidin)). Streptavidin or its homologues andbiotin enter into a stable bond, so that any biotinylated protein bindsto (particle) surfaces treated with streptavidin. The same applies iffunctionalised magnetic particles are used.

The biotinylation of proteins is known and is a standard method inbiochemistry. There is usually used for the biotinylation an activeester (for example NHS, N-hydroxysuccinimide) with which biotin iscoupled to free amino functions of a catcher protein. Biotinylatedcatcher proteins are usually bound to (magnetic) particles treated withstreptavidin or homologues thereof.

Catcher proteins which comprise protein affinity tags can be bound to a(particle) surface via metal complexes. To that end, use issubstantially made of the principle that certain amino acids form stablecomplexes with suitable metal ions (for example Co²⁺, Ni²⁺, Cu²⁺ andZn²⁺). Such amino acids can be present naturally in the protein or theycan be introduced into the protein as polypeptides, as what are known as“tags”. Common tags are Arg-tag, c-Myc-tag, FLAG-tag and His-tag. Themetal ions are bound to the (particle) surface with the aid of NTA(nitriloacetic acid), CMA (carboxymethyl aspartate) or IDA (iminoaceticacid). In the above-mentioned functionalisation, the same principles asare otherwise applied in the chromatographic purification of proteins,in particular in immobilised metal affinity chromatography (IMAC), aresubstantially used.

By using the above-mentioned principles, a person skilled in the art canreadily adapt the method according to the invention to his particularneeds in that the functionalised (magnetic) particles can individuallybe adapted to the assay to be used or to the biological analyte oranalytes to be determined.

In the case where the functionalised surfaces are already present in thedevice, the invention relates in one embodiment to the fact that theintroduction of the functionalised surfaces into the device, that is tosay step (a), is carried out as a step (a′), namely treating at leastone surface inside the device, preferably the base of the device,wherein the end of the structural element that is remote from the baseis not treated, with a coating buffer that promotes binding of thecatchers to the base, and adding at least one type of catcher, whereinthe catchers are preferably added in solution.

Accordingly, the invention relates also to a device, to a measuringchamber according to the invention and to a microplate according to theinvention in which at least one surface of the device, preferably thebase of the device, is functionalised, wherein the end of the structuralelement that is remote from the base is not functionalised. Treatedsurfaces of a device are here also referred to as “stationary” surfaces.

After addition of the catchers, it is advantageous if the coating bufferacts at the desired site provided for functionalisation for severalhours or overnight. Coating buffers suitable for populating surfaceswith catcher molecules are known. An example thereof is a basiccarbonate buffer with which, for example, the catcher molecule protein Acan be bound at a desired site, for example on the base of the device.

The functionalisation of the base of the device, of the measuringchamber according to the invention and of the microplate according tothe invention can take place by streptavidin, so that a plurality ofdifferent catchers, which have previously each been provided(conjugated) with biotin groups, are bound at the desired site.

By selecting suitable functionalised surfaces, it is possible toinfluence the test duration. In principle, a large functionalisedsurface increases the probability that the biological analyte oranalytes to be measured, and if desired and provided the luminescencemarker or markers, will come into contact with catchers on account ofdiffusion. By selecting a suitable size for the surface and the type andnumber of the catchers present thereon, the speed of the measurement canbe influenced. In order to accelerate the method according to theinvention, it is preferred to use large surfaces on which a plurality ofcatchers are situated. This applies to particle surfaces as well as tostationary surfaces.

A further factor which influences the speed of the method according tothe invention, in addition to the size of the functionalised surfaceused, is the binding capacity thereof. In the case of functionalisedparticles, their binding capacity is understood as being: capacity tobind a specific quantity of biological analyte to the functionalisedparticle in relation to the amount or weight, mass of functionalisedparticles present. A further speed-determining parameter in the methodaccording to the invention is, as described in greater detail below, thecareful mixing of the test components. It is thereby immaterial whetherthe particles are magnetic or non-magnetic.

The functionalised (magnetic) particles to be used as functionalisedsurfaces in the method according to the invention are availablecommercially or are correspondingly prepared individually for themethod.

For the preparation of such individually functionalised particles therecan be used in particular particles of synthetic or natural polymers,for example polystyrene, latex, agarose, polylactide or PMMA, as well aspolysaccharides. Particles of porous materials, such as, for example,agarose, are preferred. Particularly suitable are commercially availableparticles which are used for the chromatographic purification ofbiological analytes, for example proteins. They are conventionallysupplied in the form of a slurry. The above applies analogously tomagnetic particles, wherein the synthetic or natural polymers surroundthe magnetic core.

The non-magnetic particles which can be used according to the inventionare often spherical particles and have typical mean diameters in therange of from approximately 0.1 to approximately 200 μm. Preferredparticles according to the invention have mean diameters in the range offrom approximately 20 to approximately 200 μm, in particular fromapproximately 80 to approximately 200 μm, and do not bindnon-specifically to the test components. Particles which have a bindingcapacity of more than 200 mg IgG or analyte per ml are preferredaccording to the invention.

The magnetic particles (also called “magnetic beads”) which can be usedaccording to the invention are those which are usually used forisolating or analysing biological analytes (see, for example, WO2006/112771). The polymer coating of magnetic particles usually consistsof sugars, polyvinyl alcohol or of silicates. The magnetic particles canbe functionalised in a manner analogous to that described for theparticles or they are available commercially in the form offunctionalised magnetic particles (for example Protein A Mag-Sepharose®particles from GE Healthcare Art. No. 28-9440-06). Particularly suitablemagnetic particles according to the invention have a coating of porousmaterial, for example agarose, and have a core of ferrite or magnetite(for example Mag-Sepharose® particles).

Magnetic particles are likewise spherical particles and have typicalmean diameters in the range of from approximately 1 to approximately 100μm.

The functionalised magnetic or non-magnetic particles to be used incombination with a specific biological analyte can easily be determinedby a person skilled in the art, and in some cases such combinations arealready in use in the further developed immunoassay methods of the priorart.

The following also applies when functionalised magnetic particles areused.

In the method according to the invention, steps (a) (optionally (a′)),(b) and (c) can be carried out one after another, that is to saysequentially, or at least two of steps (a), (b) and (c) take placesimultaneously, for example by placing functionalised surfaces in thedevice, followed by the measuring solution, which contains the sampleand the luminescence markers. Alternatively, the sample containing theanalyte is placed in the device, and the luminescence marker and thefunctionalised surface in the form of functionalised particles are addedthereto. In the case of sequential addition, the sequence of the stepsis not usually important. It is possible to carry out step (b) beforestep (a) and (c), or step (b) before step (c) and (a), or step (c)before step (a) or (b). In competitive immunoassays, it is advantageousif step (b) and step (c) are carried out simultaneously. If a devicethat already has functionalised surfaces is used, step (a) is omitted.

In the method according to the invention, step (a) and step (c) arepreferably carried out, simultaneously or sequentially, before theremaining method steps. Likewise preferably, step (a) or step (c) iscarried out before the remaining method steps.

The time interval between steps (a) and/or (c) and the remaining methodsteps can be small or large (for example several hours, days, weeks,months or years). If the interval is greater than 12 hours, it isadvantageous if the test components introduced in steps (a) and/or (c),that is to say the functionalised (magnetic) particles and/orfluorescence markers that are introduced, are present in dried form inthe measuring chamber. The measuring chambers are dried at roomtemperature, preferably at an elevated temperature of not more than 37°C., preferably 35° C. The duration of the drying is generally governedby the quantity of aqueous solution introduced into the measuringchamber. Conventional drying times are in the range of from 12 to 24hours.

Before drying, further substances can optionally be introduced into themeasuring chamber, which further substances are to ensure that thefunctionality/activity of the (magnetic) particles and/or of theluminescence markers or fluorescence markers is retained after drying.Suitable substances are BSA, sugars or detergents such as Tween 20.

Accordingly, the invention relates also to the measuring chambers andmicroplates according to the invention in which there are present, ineach case in the dried state,

-   (i) at least one type of functionalised particle or functionalised    magnetic particle and at least one type of fluorescence marker, or-   (ii) at least one type of functionalised particle or functionalised    magnetic particle, or-   (iii) at least one type of fluorescence marker,

and to the use thereof in the method according to the invention.

The invention relates likewise to the production thereof. To that end,the procedure is as described above, namely by drying or freeze dryingthe corresponding test component after it has been introduced into themeasuring chamber according to the invention.

If measuring chambers are used in which at least one type offunctionalised particle or functionalised magnetic particle and/or atleast one type of fluorescence marker, in the dried state, is alreadypresent, the step or steps with which the test component in question isadded are omitted in the method according to the invention. For example,if the functionalised (magnetic) particles are already dried, theintroduction of functionalised (magnetic) particles, that is to say step(a), is omitted.

In the method according to the invention, bonds form between the testcomponents, which bonds are dependent on the affinities of the testcomponents for one another and the concentrations thereof. It isnecessary for the method according to the invention that the quantity ofunbound luminescence markers under given and constant test conditions isdependent only on the quantity of biological analyte.

In one embodiment of the invention, the luminescence marker and thecatcher each bind to the biological analyte at different sites(epitopes) (shown schematically in FIGS. 6a, 6c and 7a ).

In a further embodiment of the invention, the luminescence marker andthe biological analyte can each bind to the catcher molecule at the samesite (shown schematically in FIG. 6b ).

In a further embodiment of the invention, a primary antibody binds tothe biological analyte instead of the luminescence marker, and theluminescence marker binds to the primary antibody (shown schematicallyin FIG. 7b ).

In order to accelerate the formation of a bond, it is expedient to mixthe test components thoroughly once all the test components have beenintroduced into the device, measuring chamber or microplate according tothe invention. Mixing can take place manually or in an automated manner.The quicker the test components come into contact, the quicker theequilibrium is established and thus the biological analyte ismeasured/determined. The same applies if functionalised magneticparticles are used.

If it is desired to carry out the method more quickly, it is possible todispense with establishing equilibrium. In this case, it is important toobserve the same incubation times and conditions for all samples andcalibrators.

For automated mixing, the unit in which the device according to theinvention is situated (for example measuring chamber or microplate) isplaced onto shakers (shaking devices) which are conventional in thelaboratory. Such shaking devices are known. The optimum shaking speedcan be found by simple methods. The use of a vortex apparatus is alsoconceivable.

The aim of mixing is to increase the probability of the test componentscoming into contact with one another, so that the desired bonds form asrapidly as possible and equilibration of the binding of the componentsis achieved quickly. The time required for equilibration of the bindingof the components can vary and can easily be found. With optimal mixing,times in the range of from 15 to 30 minutes are usually required toachieve equilibration of the binding of the components that issufficient for the measurement.

Alternatively to free diffusion or shaking, the probability of the testcomponents coming into contact with one another, in particular of thebiological analyte coming into contact with a catcher molecule, can beincreased if the functionalised surfaces (preferably in the form offunctionalised particles) are introduced into fluidic systems ormicrofluidic systems. In such systems, the biological analyte oranalytes to be determined actively wash over the surfaces, so that thebiological analyte or analytes accumulate on the functionalised surface(preferably functionalised particles). Microfluidic systems are oftenused to increase the sensitivity of detection in difficult samples, forexample human serum.

Luminescence markers according to the invention can bind the desiredtest components (in particular the biological analyte or analytes) andat the same time are capable of emitting light. All conceivableluminescence markers can be used in the device according to theinvention and in the method according to the invention. Fluorescencemarkers are preferably used.

Within the scope of the present invention, the expression “boundluminescence marker” is understood as meaning all luminescence markerswhich are bound to the functionalised surfaces directly or via furthertest components (for example via analyte or a primary antibody). At thesame time, the term “unbound” luminescence or fluorescence marker refersto those markers which are not bound to the functionalised surfaceeither directly or via further test components.

Luminescence or fluorescence markers which bind to specific biologicalanalytes and/or functionalised surfaces are known and are availablecommercially. There are suitable, for example, Alexa 647- or Alexa488-conjugated antibody fragments (for example Alexa 647-conjugatedAffiniPure F(ab′)₂ fragment goat anti-human IgG F(ab′)₂ specific andpolyclonal rabbit anti-hen IgY (H+L)-Alexa Fluor 488, Alexa 488AffiniPure F(ab′)₂ fragment goat anti-human IgG, F(ab′)₂ specific) orfluorescein isothiocyanate (FITC)-conjugated polyclonal chickenanti-human IgG (H+L) antibody and R-phycoerythrin AffiniPure F(ab′)₂fragment goat anti-human IgG, Fcγ fragment specific.

Luminescence or fluorescence markers can also be produced individually.To that end, a luminescent or fluorescent dye is linked to a biologicalmolecule (for example protein, antibody, antibody fragment) which has asuitable binding site and is able to bind the desired test component(preferably the biological analyte or analytes to be determined).Linking takes place by common methods, for example by using luminescentor fluorescent dyes which are equipped with NHS ester or maleimidegroups and can thus be coupled to antibodies and proteins. The linkingcan additionally be streptavidin-mediated, in a manner similar to thatdescribed above, by binding a biotinylated protein to a luminescent orfluorescent dye equipped with streptavidin. The natural fluorescentproteins, such as, for example, GFP (green fluorescent protein) and itsderivatives, can be used as fluorescence markers either alone ortogether with other proteins, as so-called fusion proteins.

The dye and the binding site are chosen in dependence on the biologicalanalyte to be determined by the use of the device according to theinvention or by carrying out the method according to the invention, andthe assay to be used therefor.

It is possible to use all known fluorescent dyes which are otherwisealso used in measurements of biological samples. Such dyes are, forexample, cyanines, coumarins, fluoresceins, rhodamines and derivativesthereof, such as, for example, fluorescein isothiocyanate. They areavailable commercially (for example Alexa™, Dy-Light™, Atto™ orOyster™).

In addition to the above-mentioned fluorescent dyes, fluorescent quantumdots (“Qdots”) can also be used to produce fluorescence markers. Suchfluorescence markers are distinguished by particularly high fluorescenceemission.

It is conceivable that the biological analyte to be determined is itselfcoupled with a fluorescent dye and used as the fluorescence marker. Suchfluorescence markers are suitable for competitive assays in which thefluorescence marker competes with the same, non-fluorescence-labelledbiological analyte to bind to the catcher molecule.

In one embodiment of the invention, different fluorescence-labelledantibodies are used as the luminescence marker, so that differentanalytes or epitopes in different fluorescence channels can be detectedsimultaneously (multiplexing).

Unless indicated otherwise, the term “sample” is understood as meaningthe following: fluid, buffer solutions of any kind, media for cellculture and for fermentations, as well as blood, blood plasma, urine,serum, all of which contain at least one type of biological analyte.

Particularly suitable samples according to the invention are fluid,buffer solutions of any kind, media for cell culture and forfermentations.

Unless indicated otherwise, the expression “aqueous solution” isunderstood as meaning the following: An aqueous, preferably pH-bufferedsolution (for example a Tris buffer) in which (further) substancesconventional in protein biochemistry, such as, for example, bovine serumalbumin (BSA), detergents (for example Tween 20) or the like, areoptionally present.

The expression also includes reaction and binding buffers. Bindingbuffers are known and can be adapted to the particular testrequirements.

Unless indicated otherwise, the expression “measuring solution” isunderstood as meaning an aqueous solution in which the test componentsare present in solution and/or suspension while the method according tothe invention is being carried out.

Unless indicated otherwise, the expression “test component” isunderstood as meaning at least one of the following components:luminescence markers, functionalised stationary surfaces, functionalisedparticles, or functionalised magnetic particles, and sample as well ascatcher molecules and primary antibodies.

The test components can be used in the method according to the inventionin solution or suspension in a solvent, preferably in an aqueoussolution. Likewise, the test components can be used in the methodaccording to the invention in pure form, that is to say without theaddition of a solvent. This is the case, for example, with a sample whenit is fluid, buffer solutions or cell culture media, each of whichcontains at least one biological analyte. Preferred solvents are aqueoussolutions as defined herein.

Unless indicated otherwise, a biological analyte according to theinvention or the terms “analyte” and “analytes” is/are understood asmeaning a molecule which is to be determined or detected by the methodaccording to the invention. Examples of such biological analytes areproteins, antibodies, protein or antibody complexes, peptides, DNA, RNA,complexes of one or more biomolecules, viruses or one or more biologicalcells or constituents thereof, as well as low molecular weightsubstances which have biological activity or interact with biomolecules.Biological analytes which can be detected particularly well in themethod according to the invention are: proteins, antibodies, protein orantibody complexes, peptides, DNA, RNA, complexes of one or morebiomolecules, and viruses.

Unless indicated otherwise, the term “introduction” or “introduce” isunderstood as meaning the following: Manual or automatic filling of thedevice, or of the measuring chamber, by conventional measures such as,for example, pipetting or by the use of microfluidic systems.

Unless indicated otherwise, the term “binding” is understood as meaningthat at least two units come into contact with one another so that astable binding equilibrium is established between them. Such binding isgenerally mediated by interactions and results in the correspondingbonds, such as for example, hydrogen bonds, salt bonds, complex bondsand covalent bonds. The bonds utilised in the method and deviceaccording to the invention are often a mixture of several of theabove-mentioned interactions.

Luminescence within the meaning of the invention includes all knowntypes of luminescence, in particular fluorescence excited by light,bioluminescence and chemiluminescence, with fluorescence beingpreferred. In the fluorescence measurement, molecules are raised to anexcited electronic state by excitation light and then return to theground state by giving up the energy by emitting radiation (fluorescenceradiation of one or more specific wavelengths or fluorescence).Measuring the fluorescence of molecules is an established method.

Unless indicated otherwise, the expression “magnetic element” isunderstood as meaning the following: Coating or magnetic layer, such as,for example, the foil Permaftex 518 from Rheinmagnet. The layerthickness and the foil is so selected that the magnetic strength of thefoil allows the functionalised magnetic particles to sediment on thebottom of the device after mixing only outside the measuring window,while the measuring window remains free, but without interfering withthe efficient mixing of the test components (preferably on a shaker).Suitable field or magnetic strengths are thus those which are weakenough to allow the test components to be mixed and strong enough toattract the magnetic particles after mixing, whereby the measuringwindow must be kept free of magnetic particles. Suitable magnetic foilsare available commercially. The measuring windows are produced in thefoil by punching or by laser cutting.

If Protein A Mag-Sepharose® particles (GE Healthcare Art. No.28-9440-06) are used in combination with the foil Permaflex 518 fromRheinmagnet, a layer thickness of approximately 1 mm is advantageous.

Unless indicated otherwise, the following applies also to the methodsaccording to the invention in which functionalised magnetic particlesare used.

According to the invention, fluorescence markers are preferably used inthe method according to the invention and the measurement in step (d) isa fluorescence measurement and is carried out with a fluorescence meter.

The device according to the invention and the method according to theinvention allow a time-resolved, kinetic fluorescence measurement to becarried out. This is advantageous, for example, if the establishment ofthe binding/reaction equilibrium is to be monitored.

Step (e) in the method according to the invention, namely determiningthe quantity and/or concentration of the biological analyte or analytes,is carried out using a calibration series.

For the calibration series, the method according to the invention iscarried out with different, defined concentrations/quantities of theanalyte which cover the desired or expected concentration range of theanalyte, and in each case with identical test conditions, in particularthe concentration of the luminescence marker. From the measured valuesof the luminescence emission of the unbound luminescence marker there isthen produced a calibration curve, or a function, by means of which theanalyte concentrations in samples of unknown analyte content aredetermined. Examples of such calibration curves are shown in FIG. 5a to5e . The above-mentioned calibration series and the preparation ofcalibration curves for determining values are conventional inbiochemistry. A person skilled in the art knows how to prepare such acalibration curve in order to obtain meaningful results.

The method according to the invention determines the luminescence of theluminescence markers present in unbound form in solution. This is novel,because the known methods for determining biological analytes are basedon measuring the luminescence markers bound to the biological analytes.If the fluorescence of unbound luminescence markers is measured in theprior art, the measurement is mostly preceded by an enzymatic reactionor a cleavage, in which a fluorescence marker is cleaved from adetection antibody, or is produced by FRET or particulate probes. Such acleavage is not provided in the method according to the invention.

For determining the concentration of biological analytes by means of themethod according to the invention, and if it is not a competitive assay,it is advantageous if as large a proportion as possible of thebiological analytes is bound to the catcher molecules. This isfacilitated by the fact that the catcher molecules are present in excesson the surface (preferably particle surface) and have a high affinityfor the analyte. Ideally, the binding reaction of the analyte to thecatcher molecules takes place completely.

In the above-mentioned case, the concentration of the unboundluminescence marker (for example fluorescence marker) is dependent onlyon the dissociation constant K_(d) between the analyte and theluminescence marker (for example fluorescence marker), the concentrationof the luminescence marker (for example fluorescence marker) that isused, and the concentration of the biological analyte that is to bedetermined.

If the concentration of the luminescence markers (for examplefluorescence markers) is kept constant in the assay, the concentrationof the unbound luminescence markers (for example fluorescence marker)and thus the measured luminescence intensity (for example fluorescenceintensity) depends only on the concentration of the biological analyte.

For determining the concentration of the biological analytes by means ofthe method according to the invention and with a competitive assay, itis advantageous if the number of catcher molecules is smaller than orequal to the number of luminescence markers, so that each binding of ananalyte molecule leads to an increase in the number of unboundluminescence markers.

In addition to determining the concentration of the analyte, furtherinformation about the binding of the analyte to the test components, forexample the dissociation constant K_(d), or IC or EC₅₀ values, can alsobe obtained with the method according to the invention.

By means of the method according to the invention using the deviceaccording to the invention, the determination, in particular thequantification, of the biological analytes can be carried out with avery simple, namely one-stage, assay protocol. Such one-stage assays arealso called “mix-and-measure assays”. In the method according to theinvention, the test components are simply combined, and establishment ofbinding equilibrium is awaited.

After waiting for the sedimentation of the particles, the measurementcan immediately be begun. Within a short time, normally in less than onehour, the desired data are obtained, which, after correlation with thecalibration curve, yields the desired quantitative or qualitativeresult. Simple luminescence or fluorescence detection devices(fluorescence readers), as are often present in the laboratory, aresufficient for the measurement.

The method according to the invention can be configured as anon-competitive or competitive assay. Sandwich immunoassays with directand indirect detection can also be carried out with the method accordingto the invention. Preferred assays to be carried out with the methodaccording to the invention are the direct assay, sandwich assay withdirect or indirect detection, displacement assay, competitive assay andsecondary assay.

In the direct assay, the biological analyte is generally an antibodywhich is bound to a catcher molecule which is itself not an antibody butan antigen to which the antibody binds specifically with its variableportion (shown schematically in FIG. 6c ) or protein A and G which bindthe antibody at their Fc portion (shown schematically in FIG. 6a ).

In an inhibition assay, that is to say displacement assay, thebiological analyte wholly or partially prevents the luminescence markerfrom binding to the functionalised surface by binding to the bindingsite of the luminescence marker so that the binding site is no longeravailable for binding to the catcher molecules (shown schematically inFIG. 6d ).

In the sandwich assay with direct detection, the biological analyte isgenerally a protein or an antibody which is bound by the variableportion of an antibody which acts as the catcher molecule, theluminescence marker, preferably fluorescence marker, binding to a secondbinding site (epitope) of the analyte and thus forming the sandwich(shown schematically in FIG. 7a ).

In the sandwich assay with indirect detection, the biological analyte isgenerally a protein or an antibody which is bound by the variableportion of an antibody as catcher molecule, luminescence markers,preferably fluorescence markers, not binding directly to the analyte butto a further primary antibody, which binds to a second binding site(epitope) of the analyte (shown schematically in FIG. 7b ).

In all the suitable assay variants, a defined quantity of functionalisedsurfaces and luminescence marker, preferably fluorescence marker, andoptionally primary antibodies is introduced into the device or measuringchamber according to the invention.

In the competitive assay, a defined quantity of catcher molecules orfunctionalised surfaces, the sample and luminescence marker, preferablyfluorescence marker, which competes with the analyte to bind to the samebinding site (epitope) on the catcher molecules, are together introducedinto the device or measuring chamber according to the invention. Thereaction is carried out therein, with shaking, until the reactionequilibrium is established, and sedimentation of the particles isawaited. The quantity of unbound luminescence marker, preferablyfluorescence marker, in the measuring solution is then measured and theconcentration of the analyte is determined via a calibration curve orcalibration function.

The concentration of the unbound luminescence marker, preferablyfluorescence marker, is thereby dependent only on the quantity of theanalyte, which wholly or partially prevents the luminescence marker,preferably the fluorescence marker, from binding to the catchermolecules and thus effects an increase in the fluorescence signal in thedetection region.

The device according to the invention for use in the method according tothe invention is so configured that it is capable of separating testcomponents bound to functionalised surfaces and unbound test components.To that end, the device contains a structural element. The structuralelement is in particular in the form of a protrusion, the cross sectionof which can have any desired geometry (for example circular,rectangular, triangular), wherein the end that is remote from the baseis such that no test components settle there when they are particles.The structural element advantageously has a protrusion on the base ofthe device which extends straight upwards or tapers, and is at leastpartly transparent. The end can have a convex shape, for example, and/orcan have a small diameter. The structural element is preferably in theform of a cone, a truncated cone or a pyramid and protrudes from thebase of the device. The structural element can further have an opticalcomponent, for example a lens, at the end that is remote from the baseof the device. It preferably has the shape of a mandrel, cone ortruncated cone. The structural element can be made of polypropylene andis transparent. Other geometric forms of a protrusion which fulfil thepurpose described hereinbelow are conceivable.

The purpose of the structural element is to keep free the beam pathprovided in the method according to the invention for the detection ofthe luminescence and to prevent any luminescence of the boundluminescence markers that may falsify the result from reaching themeasuring device. The presence of a structural element for separation isimportant because the biological analytes are determined via theluminescence of the luminescence markers that are present in unboundform. It is at least partly transparent.

The device according to the invention likewise has a region which servesas a measuring window. The measuring window can be present in the baseof the device or at the end of the device that is opposite the base. Thebase of the at least partly transparent structural element can be ameasuring window. Excitation and detection usually take place throughthe measuring window. It is, however, also possible that the measuringwindow is used only for detection. The configuration and the position ofthe measuring window inside the device or measuring chamber can vary andbe adapted to the requirements of the measurement. It is important,however, to ensure that only the luminescence of the luminescencemarkers present in unbound form is measured through the measuringwindow. The sensitivity of the method in which the device is used can bemodulated by suitably selecting the size of the measuring window and thetype of luminescence marker.

The region within the device or measuring chamber in which theluminescence markers emit luminescence and which can beobserved/measured spectroscopically through the measuring window iscalled the “measuring region” or “detection region” hereinbelow.

The region within the device or measuring chamber which cannot beobserved/measured spectroscopically through the measuring window isreferred to as the “separation region” hereinbelow.

In the device according to the invention, the detection region and theseparation region are connected, so that the test components caneffectively and easily be exchanged between the two regions. Theconcentration of the unbound luminescence marker is thus the samethroughout the device/measuring chamber.

In a preferred embodiment of the invention, the measuring window isoptically connected to the end of the structural element that is remotefrom the base and represents the base area of the structural element.For example in that the structural element constitutes a protrusion onthe base of the device which extends straight upwards or tapers and istransparent, and the base of the device has an opaque layer or coating,the base area of the structural element not being coated. The opaquelayer (for example a black lacquer layer) is so applied that a region onthe base of the device that is situated beneath the structural elementis kept free, whereby the measuring window is formed.

If light is then directed into the measuring chamber from the base ofthe device, the radiation runs through the structural element into themeasuring region. After excitation of the luminescence markers in themeasuring region, the emission can be measured again at the measuringwindow.

In a further embodiment of the method there can be used a device,measuring chamber or microplate according to the invention, inparticular as described above or in one of embodiments [0], [A], [B],[C], [D], [E], [F] or [G], that does not have an opaque layer, namelywhen the fluorescence meter used is a fluorescence microscope in whichthe measuring optics is so adapted that only the fluorescence emissionfrom the detection region is detected, for example by choosing an opticswith suitable imaging properties and a suitable collection efficiency(for example automated fluorescence microscope of type NyONE (SynenTec,Elmshorn, Germany) incl. 10× objective from Olympus).

If functionalised particles are used as the functionalised surfaces inthe device according to the invention and in the method according to theinvention, separation of the unbound luminescence markers fromparticle-bound luminescence markers usually takes place bysedimentation. To that end, the device loaded with measuring solution isnot moved for a certain time. The functionalised particles withluminescence markers bound thereto have a higher density than thesurrounding measuring solution and sediment. The unbound luminescencemarkers remain in solution. The bound luminescence markers then settleon the base of the device around the structural element, so that theyare situated in the separation region. If functionalised (magnetic)particles are used in the method according to the invention, itaccordingly comprises also step (c′), namely allowing the device tostand for a certain time, preferably for from 1 to 30 minutes or from 1to 20 or from 1 to 15 minutes, particularly preferably for from 1 to 10minutes, or, when magnetic particles are used, for from 1 to 5 minutes,particularly preferably for from 1 to 2 minutes.

Generally, if functionalised magnetic particles are used, thesedimentation time of the bound luminescence markers is less than 5minutes. If magnetic particles are not used, the sedimentation time isless than 30 minutes, usually in the range of from 5 to 20 minutes.

If it is desired to perform a test quickly, it is advantageous to usefunctionalised particles which have a high density. In the case ofnon-magnetic particles, an average diameter of approximately 90 μm hasbeen found to be advantageous.

In this connection, high density means that the (magnetic) particlesused have a higher density than the aqueous test solution.

If non-magnetic particles which are smaller than 1 μm are used in themethod according to the invention, it is advantageous, instead ofwaiting for the particles to sediment, to insert a centrifugation stepbefore the luminescence measurement, whereby the particles are broughtonto the base of the device.

It is likewise advantageous for performing a test quickly if the(magnetic) functionalised particles have a large surface area, as aresult of which there is a high probability that a biological analytewill come into contact with a catcher molecule as it diffuses throughthe solution. The probability is increased by shaking (agitating) thedevice.

The device can be combined with known laboratory devices andconsumables, for example with microplates and Eppendorf test tubes, orother vessels and plates, in order to be able to carry out the methodaccording to the invention therewith. The side walls of theabove-mentioned equipment then form, together with the device accordingto the invention, a measuring chamber. Advantageously, a plurality ofmeasuring chambers of the same type (for example in the form of amicroplate) are accommodated in a holder system, it being advantageousif the dimensions comply with the standard of the “Society forBiomolecular Screening” (SBS) of 127.76 mm×85.48 mm×14.35 mm, in orderto be able to work on devices which are conventional in the laboratoryand thus to be able to achieve a high degree of automation.

In one embodiment of the invention, the invention relates to a measuringchamber, consisting of a chamber which is open or closed at the top,which has side walls and the base of which is formed by the device. Themeasuring chamber can have round or straight side walls. It is, however,also possible that the measuring chamber has a plurality of bases,namely when the base of the device is present in addition to the base ofthe measuring chamber.

The side walls of the measuring chamber and the base of the device, orthe side walls of the measuring chamber and another base on which thedevice is situated, are preferably connected, for example bonded orwelded, in such a manner that they are impermeable to liquids.

In a further embodiment of the invention, the device is introduced intothe well of a microplate, the edges of the well then forming the sideedges of the measuring chamber.

In a further embodiment of the invention, the invention relates to amicroplate in which the original bottom of the microplate has beenreplaced by a one-piece base containing the structural elements, thatbase, apart from the base area of the structural elements, being coatedin such a manner that it is opaque (for example by adhesive bonding ofan opaque foil with corresponding apertures, or by painting).

The production of the device in the above-mentioned SBS format iscarried out, for example, by using a microplate without a bottom andapplying in place of the bottom a transparent and non-fluorescent foil(for example of polypropylene), in which the structural elements areembossed in the arrangement corresponding to the SBS format. Such foilsrepresent a plurality of one-piece devices. The structural elements thenpoint into the openings in the microplate. Such embossed foils areavailable commercially (for example Arraytape™ from Douglas Scientific).

Application of the foil to the side of the microplate on which thebottom would actually be situated is usually carried out by adhesivebonding with an adhesive which is capable of bonding two differentplastics materials. Most simply, a UV-curing adhesive which is notfluorescent is used for that purpose. Finally, an opaque non-fluorescentlacquer layer is applied to the underside of the plastics foil in such amanner that the underside is kept free in the region of the structuralelements. It is also conceivable that the lacquer layer is first appliedto the foil and then the foil is adhesively bonded to the microplate.

The production of the microplates with magnetic elements is carried outby bonding a commercial microplate having a transparent bottom (alsocalled a “base” in this context) in an optionally reversible, that is tosay permanently connected or detachable manner, to a magnetic foil of asuitable magnetic strength, which foil has an aperture for each well(which corresponds to the measuring chamber) of the microplate, whichaperture is used as the measuring window. The apertures are so arrangedthat, when the foil is bonded to the bottom of the microplate, they aresituated centrally beneath the wells of the microplate. The optimal sizeof the aperture depends on the size of the well and can be determined bya person skilled in the art on the basis of the following example. Thediameters of the apertures must correspond to the dimensions of thewells and the number and size of the particles which are used in theassay. In the case of a 384-well microplate, apertures having a diameterof approximately 2.5 mm have been found to be advantageous in the caseof the use of approximately 1000 magnetic particles per well. Thiscorresponds to approximately half the bottom area of a commercial384-well plate and on the one hand provides a sufficient surface forseparation of the magnetic particles and at the same time provides asufficiently large measuring window for the fluorescence measurement.

Owing to the particular configuration of the device or of the measuringchamber, the bound luminescence markers or fluorescence markers, whichsediment and thus migrate out of the detection region, are not excitedto luminescence or fluorescence. The invention will be explained withreference to the examples depicted hereinbelow in the figures anddescribed in the following, without limiting the invention thereto.

FIG. 1a : Vertical section through a measuring chamber (101) having astructural element.

FIG. 1a shows a measuring chamber (101) consisting of a one-piece device(102) having a structural element (103) which has a measuring window(104) through which excitation light (105) passes into the detectionregion (106) and through which the emission of the luminescence (107) ismeasured with a suitable detection device, which at the same time alsosupplies the excitation light, or detection unit. The one-piece device(102) is connected to the side walls (108) in a liquid-impermeablemanner. On the lower side of the base of the device there is an opaquelayer (109) (for example lacquer or foil), the base area of thestructural element (110) being kept free.

FIG. 1b : Vertical section through a sequence of devices as can beintegrated, for example, into a microplate.

FIG. 1b shows a plurality of devices (102) which are introduced into thecups/wells of a microplate, the side walls (108) belonging to themicroplate and the device being a foil (111) into which the structuralelements (103) are embossed.

FIG. 1c : Vertical section through a measuring chamber (101) having amagnetic element.

FIG. 1c shows a measuring chamber (101), open at the top, consisting ofa transparent base (112), there being situated beneath the base anopaque magnetic element (here: an opaque magnetic layer or foil) (113)having an aperture (114), the aperture (114) forming the measuringwindow (104). Excitation light (105) passes through the measuring window(104) into the measuring chamber from beneath (that is to say from thebase of the device), causing emission of the luminescence (107) of theunbound luminescence markers, which is then detected through themeasuring window (104) beneath the device by means of the detection unit(115). Advantageously, and as shown, the detection unit (for example afluorescence reader) is able to emit excitation light and detect theluminescence. The base (112) is connected to the side walls (108) and tothe magnetic element (113) removably or non-removably. The base and theside walls are made of the same or different materials.

FIG. 1d : Vertical section through a sequence of measuring chambers(101) (for example in the form of a microplate).

FIG. 1d shows a plurality of measuring chambers (101) as well as thetransparent base (112) and the opaque magnetic layer or foil (113) withan aperture (114), which serve as measuring windows (104). Excitation inthe method according to the invention and also detection take placethrough the measuring window from beneath, that is to say from beneaththe base of the plurality of measuring chambers, or microplate.

FIG. 2a : Vertical section through the measuring chamber of FIG. 1a withmeasuring solution, after filling of the device but beforebinding—schematic representation.

FIG. 2a shows the measuring chamber from FIG. 1a filled with measuringsolution (201), comprising the following test components: biologicalanalyte (204), functionalised particles (203) and luminescence markers(202) immediately after introduction, homogeneously distributed and inthe unbound state before contacting by mixing. The functionalisedparticles can also be magnetic (not depicted here).

FIG. 2b : Vertical section through the measuring chamber of FIG. 1a withmeasuring solution, after filling of the device, binding andseparation—schematic representation.

FIG. 2b shows the measuring chamber from FIG. 2a filled with measuringsolution (201), comprising the test components (202), (203), (204) afterbinding and separation of the bound luminescence markers from theunbound luminescence markers. The unbound luminescence markers (202 u)are homogeneously distributed in the measuring chamber, while the boundtest components (202, 203, 204) are in the separation region (205).

FIG. 2c : Vertical section through the measuring chamber of FIG. 1c withmeasuring solution, after filling of the device but beforebinding—schematic representation.

FIG. 2c shows the measuring chamber of FIG. 1c filled with measuringsolution (201), comprising the following test components: biologicalanalyte (204), functionalised magnetic particles (203-M) andfluorescence markers (202-F) immediately after introduction,homogeneously distributed and in the unbound state before contacting bymixing.

FIG. 2d : Vertical section through the measuring chamber of FIG. 1c withmeasuring solution after filling of the device, binding andseparation—schematic representation.

FIG. 2d shows the measuring chamber of FIG. 2c filled with measuringsolution (201), comprising the test components (202-F), (203-M), (204)after binding and separation of the bound fluorescence markers from theunbound fluorescence markers. The unbound fluorescence markers (202 u-F)are distributed homogeneously in the measuring chamber, while the boundtest components (202-F, 203-M, 204) are in the separation region (205).

FIG. 3a : Top view into the measuring chamber of FIG. 2b after fillingof the device, binding and separation—schematic representation.

FIG. 3a is a top view of the measuring chamber of FIG. 2b . The boundtest components (301) are situated on the base of the device/measuringchamber. The base of the device is provided with an opaque layer (302),the base area of the structural element (303) being kept free. Theaperture at the same time represents the measuring window (305). Alsodepicted is the end (304) that is remote from the base of the device,which end is optically connected to the measuring window (305). Thestructural element is here an upwardly tapering protrusion with a roundcross section.

FIG. 3b : Top view into the measuring chamber of FIG. 2d after fillingof the device, binding and separation—schematic representation.

FIG. 3b is a top view of the measuring chamber of FIG. 2d . The boundtest components (301) are situated on the base of the measuring chamber.The base of the measuring chamber is provided with an opaque magneticlayer or foil (306) with an aperture (307). The aperture at the sametime represents the measuring window (305).

FIG. 4: Top view of a 384-well microplate (401) containing a pluralityof measuring chambers (402).

FIG. 5a : Calibration curve from Example A.

FIG. 5b : Calibration curve from Example B.

FIG. Sc: Calibration curve from Example C.

In FIG. 5a, 5b and 5c , the analyte concentration in μg/ml isrepresented on the x-axis and the fluorescence intensity is representedon the y-axis. A function is drawn (dotted line) through the measuredvalues (as diamonds).

FIG. 5d : Measurement with two fluorescence markers from Example D.

In FIG. 5d , the analyte concentration in μg/ml is represented on thex-axis and the fluorescence intensity for two fluorescence markers whichbind to different epitopes of the same analyte is represented on they-axis. The fluorescence markers emit at two different wavelengths (535and 580 nm). The corresponding functions are drawn (dotted line) throughthe measured values (each as diamonds).

FIG. 5e : Measurement of an assay in a microplate without a transparentlayer from Example E.

In FIG. 5e , the analyte concentration in μg/ml is represented on thex-axis and the fluorescence intensity is represented on the y-axis. Afunction is drawn (dotted line) through the measured values (asdiamonds). Since this example was measured with a fluorescencemicroscope, the scaling of the y-axis is different than in the precedingexamples.

FIG. 6a : Schematic representation of the binding behaviour of the testcomponents in a direct immunoassay for antibodies having a catchermolecule which binds the Fc portion of the antibody.

FIG. 6a shows a functionalised particle (601), or functionalisedmagnetic particle (601-M), consisting of a particle (602), or magneticparticle (602-M), on which a plurality of catcher molecules (603) arepresent. The biological analytes (604) bind to the catcher molecules(603) and the luminescence marker (607), or fluorescence marker (607-F).Also present are unbound luminescence markers (607 u), or unboundfluorescence markers (607 u-F). The luminescence marker, or fluorescencemarker, has a binding site (606) and a luminescent dye (605), orfluorescent dye (605-F).

FIG. 6b : Schematic representation of the binding behaviour of the testcomponents in a competitive immunoassay.

FIG. 6b shows a functionalised particle (601), or functionalisedmagnetic particle (601-M), consisting of a particle (602), or magneticparticle (602-M), on which a plurality of catcher molecules (603) arepresent. The catcher molecules (603) bind either to the biologicalanalyte (604) or to the luminescence marker (607), or fluorescent marker(607-F), the biological analyte (604) being capable of displacing thebound luminescence marker, or bound fluorescence marker, from thecatcher.

FIG. 6c : Schematic representation of the binding behaviour of the testcomponents in a direct immunoassay for antibodies having an antigen ascatcher molecule, to which the variable portion of the antibody binds.

FIG. 6c shows a functionalised particle (601), or functionalisedmagnetic particle (601-M), consisting of a particle (602), or magneticparticle (602-M), on which a plurality of catcher molecules (603) arepresent. The biological analytes (604) bind to the catcher molecules(603) and the luminescence marker (607), or fluorescence marker (607-F).Also present are unbound luminescence markers (607 u), or unboundfluorescence markers (607 u-F). The luminescence marker has a bindingsite (606) and a luminescent dye (605), or fluorescent dye (605-F).

FIG. 6d : Schematic representation of the binding behaviour of the testcomponents in an inhibition assay for antibodies.

FIG. 6d shows a functionalised particle (601), or functionalisedmagnetic particle (601-M), consisting of a particle (602), or magneticparticle (602-M), on which a plurality of catcher molecules (603) arepresent, the catcher molecules (603) being able to bind the fluorescencemarker (607-F), consisting of binding site (606) and fluorescent dye(605-F). The biological analyte (604) binds to the fluorescence marker(607-F) and thereby prevents (inhibits) binding of the fluorescencemarker to the catchers (603). In this type of binding, the complex (608,608-F) is detected, namely the luminescence marker (607), orfluorescence marker (607-F), bound to the biological analyte (604).

FIGS. 7a and 7b : Schematic representation of the binding behaviour ofthe test components in a sandwich immunoassay with direct detection(FIG. 7a ) and indirect detection (FIG. 7b ).

Both figures show a functionalised particle (701), or functionalisedmagnetic particle (701-M), which consists of a particle (702), ormagnetic particle (702-M), to which a plurality of catcher molecules(703) are bound. The catcher molecule is formed by a suitable protein(705), for example biotinylated antibody, which is bound to the particle(702), or magnetic particle (702-M), via a linker (704) (for examplestreptavidin). The protein (705) binds the biological analyte (709).Also depicted are bound luminescence markers (706), or boundfluorescence markers (706-F), and unbound luminescence markers (706 u),or unbound fluorescence markers (706 u-F). The luminescence marker(706), or fluorescence marker (706-F), has a binding site (707) and aluminescent dye (708), or fluorescent dye (708-F).

In FIG. 7b there is present, in addition to the test components shown inFIG. 7a , additionally also a primary antibody (710) which binds to theanalyte (709) and to which the luminescence marker (706), orfluorescence marker (706-F), binds.

The method according to the invention can be carried out using themeasuring chamber according to the invention as shown in FIG. 1a asfollows:

A measuring solution (201) which comprises biological analytes (204),functionalised particles (203) and unbound luminescence markers (202) isintroduced into the measuring chamber (101) (see FIG. 2a ). Ahomogeneous mixture forms. After the corresponding bonds have formed (byincubation and shaking), the bound test components sediment into theseparation region (205), where they collect (see FIGS. 2b and 3a ). Theunbound luminescence markers (202 u) can now be measured in thedetection region (105). The opaque layer (109) ensures both that thebound luminescence markers situated in the separation region cannot beexcited to emission by excitation light and that the emission light ofthe bound luminescence markers cannot reach the detection unit/devicefrom the measuring chamber through the measuring window.

The method according to the invention can be carried out using themeasuring chamber according to the invention having magnetic elements,as shown in FIG. 1c , as follows:

A measuring solution (201) which comprises biological analytes (204),functionalised magnetic particles (203-M) and unbound fluorescencemarkers (202-F) is introduced into the measuring chamber (101) (see FIG.2c ). A homogeneous mixture forms. After the corresponding bonds haveformed (by incubation and shaking), the bound test components, directedby the magnetic field of the magnetic element, sediment into theseparation region (205), where they collect (see FIGS. 2d and 3b ). Theunbound fluorescence markers (202 u-F) can now be measured in thedetection region. The opaque magnetic layer or foil (113) ensures boththat the bound fluorescence markers (202-F) situated in the separationregion cannot be excited to emission by the excitation light and thatthe emission light of the bound fluorescence markers (202-F) cannotreach the detection device (115) from the measuring chamber through themeasuring window (104).

Examples of calibrations for the method according to the invention whichare carried out are described below. The determination of the biologicalanalyte used in the calibration series takes place analogously tofollowing examples on samples of unknown analyte content, the analyteconcentration being determined via the measured fluorescence intensityof the unbound fluorescence markers using the respective calibrationcurve.

EXAMPLE A: MEASUREMENT OF A CALIBRATION SERIES IN THE DIRECT ASSAYVARIANT

The following microplate according to the invention is used for themeasurement: A black microplate (Greiner BioONE, Art. No. 781000-06)with 384 wells (measuring chambers), in each of which there is situateda 1.6 mm high conical structural element of polypropylene which tapersupwards and has a round cross section and which has a diameter of 1 mmon the side that is remote from the base. The measuring chambers areprovided on their underside with an opaque lacquer layer, wherein thelayer is not applied to the base area of the structural element so thata measuring window having a diameter of approximately 2 mm is formed onthe base.

There are used as the functionalised particles Protein A-Sepharose® 4B,Fast Flow beads (Sigma-Aldrich, Art. No. P9424) with an average diameterof 90 μm.

There is used as the fluorescence marker an Alexa 647-conjugatedantibody fragment (Jackson Immuno Research, AffiniPure F(ab′)₂ FragmentGoat Anti-Human IgG F(ab′)₂ specific Art No. 109-606-097).

The following buffer is used as the aqueous solution: 10 mM Tris, 150 mMNaCl, 0.1% bovine serum albumin (BSA), 0.05% Polysorbate 20 (Tween 20™),pH 7.4 in distilled water (called buffer hereinbelow). The recombinantlyproduced antibody rituximab (Mabthera™) is used as the biologicalanalyte for the calibration.

In the microplate, a calibration curve is prepared with a total of 12different analyte concentrations. The analyte concentrations are0/0.001/0.0033/0.0067/0.01/0.03/0.67/0.1/0.33/0.67/1.0 and 3.33 μg/mlrituximab (Mabthera™).

The procedure is as follows: 54 μl of a stock solution comprising 15 μlof Protein A Sepharose beads slurry and 60 μl of fluorescence marker (10μg/ml) in 810 μl of buffer are added to each measuring chamber.

From a concentrated stock solution of the analyte (10 mg/m), dilutionsof from 1:300 to 1:1,000,000 in buffer are prepared and [aliquots of] ineach case 6 μl thereof are added to the measuring chambers in order toachieve the target concentrations of analyte for the calibration series.

The entire microplate is shaken at 1400 rpm on an Eppendorf ThermomixerComfort for 45 minutes at room temperature. The microplate is thenremoved from the thermomixer, and a period of 5 minutes is allowed toelapse until the particles have sedimented.

The fluorescence intensity in each measuring chamber is then measuredfrom beneath (bottom reading) in a Tecan Infinite M100 fluorescenceplate reader at an excitation wavelength of 645 nm and an emissionwavelength of 675 nm.

The values obtained for the fluorescence intensity are plotted againstthe analyte concentrations and fitted using a 4 parameter fit in orderto obtain the calibration function.

The corresponding calibration curve is shown in FIG. 5 a.

EXAMPLE B: MEASUREMENT OF A CALIBRATION SERIES IN A COMPETITIVE ASSAY

The following microplate according to the invention is used for themeasurement: A black microplate (Greiner BioONE, Art. No. 781000-06)with 384 wells (measuring chambers), in each of which there is situateda 1.6 mm high conical structural element of polypropylene which tapersupwards and has a round cross section and which has a diameter of 1 mmon the side that is remote from the base. The measuring chambers areprovided on their underside with an opaque lacquer layer, wherein thelayer is not applied to the base area of the structural element so thata measuring window having a diameter of approximately 2 mm is formed onthe base.

There are used as the functionalised particles StreptavidinMag-Sepharose® particles (VWR, Art. No. 28-9857-38) having an averagediameter of 70 μm, which are functionalised with a biotinylated proteinA fragment (Affibody, Art. No. 10.0623.02.00005). The particles arefunctionalised by incubating 100 μl of particle suspension for half anhour with 0.3 μl of a solution of the biotinylated protein A fragmenthaving a concentration of 1 mg/ml and then washing the particles withbuffer.

There is used as the fluorescence marker an Alexa 488-conjugatedpolyclonal rabbit anti-chicken IgY (H+L)-Alexa Fluor 488 (Jackson ImmunoResearch, Art. No. 303-545-003).

The following buffer is used as the aqueous solution: 10 mM Tris, 150 mMNaCl, 0.1% bovine serum albumin (BSA), 0.05% Polysorbate 20 (Tween™ 20),pH 7.4 in distilled water (called buffer hereinbelow). The recombinantlyproduced antibody rituximab (Mabthera™) is used as the biologicalanalyte for the calibration.

In the microplate, a calibration curve is prepared with a total of 14different analyte concentrations. The analyte concentrations are0.001/0.0033//0.01/0.03/0.67/0.1/0.33/0.67/1.0/3.33/6.67/10/20 and 100μg/ml rituximab (Mabthera™).

The procedure is as follows: Loading the measuring chamber withfunctionalised particles by adding to each measuring chamber 48 μl of astock solution comprising 80 μl of a suspension of the above-mentionedfunctionalised particles in 1840 μl of buffer.

Preparing mixtures of fluorescence marker in each case in equalconcentrations and biological analyte in different concentrations bypreparing from a concentrated stock solution of the analyte (10 mg/ml)dilutions of from 1:10 to 1:1,000,000 in buffer. 6 μl aliquots of eachof the dilutions are combined with 6 μl of the rabbit anti-chicken IgY(H+L)-Alexa Fluor 488 antibody (from a stock solution of 10 μg/ml) andmixed.

Introducing the mixture of fluorescence marker and biological analyteinto the measuring chamber. Mixing the test components by shaking themicroplate at 1600 rpm on an Eppendorf MixMate for 30 minutes at roomtemperature. When the shaking is ended, the microplate is removed fromthe shaker, and sedimentation of the particles is awaited, which heretook approximately from 1 to 2 minutes.

Transferring the plate to a fluorescence recording device which issuitable for bottom reading (for example Tecan Safire MonochromaticFluorescence reader), in which each measuring chamber is illuminatedfrom beneath at a certain excitation wavelength (here 488 nm) and theresulting fluorescence emission or fluorescence intensity is recorded ata certain emission wavelength (here 535 nm).

Plotting the values obtained for the fluorescence intensity against theanalyte concentrations and fitting using a 4 parameter fit in order toobtain the calibration function. The corresponding calibration curve isshown in FIG. 5 b.

EXAMPLE C: MEASUREMENT OF A CALIBRATION SERIES IN A MICROPLATE HAVING AMAGNETIC ELEMENT

The following microplate according to the invention is used for themeasurement: A black microplate having a transparent bottom (GreinerBoONE, Art. No. 781091) with 384 wells, beneath which a magnetic foilhaving 384 apertures is adhesively bonded so that each aperture iscentred beneath a well of the microplate. The permanently magnetic foil(Permaflex 518, Rheinmagnet) has a thickness of 1 mm and the apertureshave a diameter of 2.5 mm.

There are used as the functionalised magnetic particles Protein AMag-Sepharose® particles (GE Healthcare Art. No. 28-9440-06) having adiameter of 37-100 μm.

There is used as the fluorescence marker a fluorescein isothiocyanate(FITC)-conjugated polyclonal chicken anti-human IgG (H+L) antibody(Abcam, Art. No. 112453).

The following buffer is used as the aqueous solution: 10 mM Tris, 150 mMNaCl, 0.1% bovine serum albumin (BSA), 0.05% Polysorbate 20 (Tween™ 20),pH 7.4 in distilled water. The antibody rituximab (Mabthera™) is used asthe biological analyte.

The assay was carried out as described in Example A, the following stocksolution comprising particles and fluorescence marker being used: 75 μlof a suspension of Protein A Mag-Sepharose® particles and 1.86 μl of thefluorescence marker in 1782 μl of buffer.

47 μl of the suspension were incubated for 30 minutes on a VariomagMonoshake (H+P) shaker with in each case 3 μl of a sample from thecalibration series of rituximab. The microplate was then allowed tostand for 5 minutes in order to await sedimentation of the particles,and the measurement was then carried out.

Measurement is carried out in a Tecan Safire fluorescence reader at anexcitation wavelength of 490 nm and an emission wavelength of 535 nm.

The calibration function is determined as described in Example A, andthe corresponding calibration curve is shown in FIG. 5 c.

EXAMPLE D: MEASUREMENT WITH TWO FLUORESCENCE MARKERS

The following microplate according to the invention is used for themeasurement: A black microplate (Greiner BioONE, Art. No. 781000-06)with 384 wells (measuring chambers) and a one-piece bottom containingstructural elements, which was produced by thermoforming. The structuralelements have the shape of a four-sided square-based pyramid. Themeasuring chambers are provided on their underside with an opaquelacquer layer, wherein the layer is not applied to the square base areaof the structural element so that a measuring window having a diameterof approximately 2.5 mm is formed on the base.

This assay is carried out using particles which were placed in themicroplates in the already dried state. To that end, 30 μl of asuspension of Protein A-Sepharose® 4B Fast Flow beads are dried on themicroplate at 35 degrees Celsius. A stock solution of 1.6 μl of a firstfluorescence marker, namely Alexa 488 AffiniPure F(ab′)₂ Fragment GoatAnti-Human IgG, F(ab′)₂ specific (Jackson Immuno Research Art. No.109-546-097), and 3.93 μl of a second fluorescence marker, namelyR-Phycoerythrin AffiniPure F(ab′)₂ Fragment Goat Anti-Human IgG, FcγFragment specific (Jackson Immuno Research Art. No. 109-116-170), in1607 μl of buffer are prepared, and in each case 54 μl thereof areincubated for 30 minutes on a Vario-mag Monoshake shaker (H+P) with ineach case 6 μl of a sample from the calibration series of rituximab(Mabthera™). The microplate was then allowed to stand for 15 minutes inorder to await sedimentation of the particles, and the measurement wasthen carried out.

The following buffer is used as the aqueous solution: 10 mM Tris, 150 mMNaCl, 0.1% bovine serum albumin (BSA), 0.05% Polysorbate 20 (Tween™ 20),pH 7.4 in distilled water.

The measurement is carried out in a Tecan Safire fluorescence reader atan excitation wavelength of 490 nm and at emission wavelengths of 535 nmfor detection of the first fluorescence marker (=Alexa 647-conjugatedantibody fragment (Jackson Immuno Research, AffiniPure F(ab′)₂ FragmentGoat Anti-Human IgG F(ab′) specific 488) and 580 nm for detection of thesecond fluorescence marker (=R-Phycoerythrin).

The respective calibration functions are determined analogously toExamples A and B. The corresponding calibration curve is shown in FIG.5d . FIG. 5d further shows that the simultaneous detection of differentbinding sites on the analyte (here: rituximab (Mabthera™)) is possiblewith similar calibration curves.

EXAMPLE E: MEASUREMENT OF AN ASSAY IN A MICROPLATE WITHOUT AN OPAQUELAVER

The measurement is carried out using the microplate described in ExampleD with structural elements but without the opaque lacquer layer.

The following buffer is used as the aqueous solution: 10 mM Tris, 150 mMNaCl, 0.1% bovine serum albumin (BSA), 0.05% Polysorbate 20 (Tween™ 20),pH 7.4 in distilled water.

The assay is carried out according to Example A using the followingstock solution comprising functionalised particles and fluorescencemarker: 600 μl of a pre-diluted suspension of Protein A-Sepharose® 4BFast Flow beads and 1.06 μl of the fluorescence marker Alexa488AffiniPure F(ab′)₂ Fragment Goat Anti-Human IgG, F(ab′)₂ specific(Jackson Immuno Research Art. No. 109-546-097) in 478 μl of buffer. Ineach case 54 μl thereof are incubated for 30 minutes as described inExample A on a Variomag Monoshake shaker (H+P) with in each case 6 μl ofa sample from the calibration series of rituximab (Mabthera™). Themicroplate is then allowed to stand for 15 minutes to awaitsedimentation of the particles, and the measurement is then carried out.

An automated fluorescence microscope of type NyONE (SynenTec, Elmshom,Germany) is used for the measurement. With a 10× objective from Olympus,an image positioned centrally in the middle of the well is recorded ineach case and the fluorescence intensity in the image is measured.

A calibration function is determined analogously to Example A. Thecorresponding calibration curve is shown in FIG. 5 e.

1. A measuring chamber for quantitatively determining biologicalanalytes in an aqueous solution in the presence of one or morefunctionalised surfaces, comprising a device and at least one side wallsurrounding the device, wherein the base of the device can form the baseof the measuring chamber and wherein the device has an at least partlytransparent structural element, and wherein the base of the device,apart from the base area of the structural element, is opaque.
 2. Themeasuring chamber according to claim 1, wherein the structural elementis in the form of an upwardly tapering protrusion, the cross section ofwhich can have any desired geometry, wherein the end of the structuralelement that is remote from the base is such that no test componentssettle there.
 3. The measuring chamber according to claim 1, wherein thebase of the device forms the base of the measuring chamber or whereinthe base of the device is not the base of the measuring chamber but afurther base is present.
 4. The measuring chamber according to claim 1,wherein there is a permanently connected or removable opaque magneticlayer on the base of the measuring chamber, which layer has atransparent measuring window.
 5. A microplate for quantitativelydetermining biological analytes in an aqueous solution in the presenceof one or more functionalised surfaces, wherein the microplate comprisesat least one measuring chamber comprising a device and at least one sidewall surrounding the device, wherein the base of the device can form thebase of the measuring chamber, wherein the device has an at least partlytransparent structural element, and wherein the base of the device,apart from the base area of the structural element, is opaque.
 6. Themicroplate according to claim 5, wherein the structural element is inthe form of an upwardly tapering protrusion, the cross section of whichcan have any desired geometry, wherein the end of the structural elementthat is remote from the base is such that no test components settlethere.
 7. A microplate for quantitatively determining biologicalanalytes in an aqueous solution in the presence of one or morefunctionalised surfaces, wherein there is introduced into at least onewell of the microplate a device which has an at least partly transparentstructural element which is in the form of an upwardly taperingprotrusion and the cross section of which can have any desired geometry,wherein the end of the structural element that is remote from the baseis such that substantially no test components settle there, and whereinthe edges of the well form the side walls of a measuring chamber.
 8. Themicroplate according to claim 7, wherein the base of the device, apartfrom the base area of the structural element, is opaque.
 9. Themicroplate according to claim 7, wherein the original bottom of themicroplate is replaced by a one-piece base having structural elements,wherein a structural element is situated in each of the wells of themicroplate, and wherein the base, apart from the base area of thestructural elements, is provided with an opaque coating.
 10. Themicroplate according to claim 7, wherein functionalised surfaces orfunctionalised particles and or fluorescence markers in dried form aresituated in at least one of the measuring chambers.
 11. A kit forquantitatively determining biological analytes in an aqueous solution inthe presence of one or more functionalised surfaces, comprising themicroplate according to claim 7, and further comprising at least onetype of functionalised surfaces or particles, at least one type offluorescence marker, and a reaction buffer.
 12. A kit for quantitativelydetermining biological analytes in an aqueous solution in the presenceof one or more functionalised surfaces, comprising the microplateaccording to claim 9 and a reaction buffer.
 13. The microplate accordingto claim 7, wherein the measuring chamber has a detection region whichis accessible to light through the bottom of the measuring chamber,wherein the structural element is transparent, wherein the end of thestructural element that is remote from the base is such that no testcomponents settle there, and wherein fluorescence emission of unboundfluorescence markers in the detection region is measured with afluorescence reader whose optical configuration is such that it is ableto measure only the fluorescence emission in the detection region. 14.The microplate according to claim 13, wherein an original bottom of themicroplate is replaced by a one-piece base containing the structuralelements.
 15. The microplate according to claim 13, wherein thefluorescence reader is a fluorescence microscope.